Shoot Regeneration Is Not a Single Cell Event

Abstract

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

Florigen governs shoot regeneration

“…Reducing the amount of NO in Arabidopsis plants promoted flowering, increasing NO inhibited flowering 37 , as was shown here. In parallel, reducing the amount of NO decreases regeneration, and increasing the amount of cellular NO increases regeneration 30 (and in this study). We postulated a connection between NO and FT gene family members; it seems that NO level controls FT mRNA expression and thus flowering and regeneration ability.…”

“…Treating tobacco seedlings with DiPhenyl that inhibits NO synthesis 29 , 30 shows a similar phenotype of lacking NO as in Arabidopsis nos1 mutant 31 (Fig. 2 d) i.e., impaired growth, yellowish first true leaves, reduces root size, defective abscisic acid-induced stomatal movements, and most importantly, induces early blooming 31 .…”

“…Ongoing analysis is the effect of cytokinins and auxin combination on shoot regeneration in mature vs. juvenile leaves. Knox gene family was implicated in FT function 27 and in shoot regeneration 30 . We are currently testing the effect of Knox gene family knockout using crisper technology on shoot regeneration.…”

“…Plants were grown in sterile containers in a growth room with 16 hours of light and eight hours of darkness at 26 °C for weeks until leaves were ready to be harvested for regeneration. Tobacco plants were transformed as described before 30 , 44 with Avocado ( Persea Americana ), FLOWERING LOCUS T-like plasmids obtained from Ziv et al 45 , pear ( Pyrus communis ) from Frieman et al 21 , and pepper ( Capsicum annum ) from Borovsky et al 46 . Transgenic tomato ( Solanum lycopersicon M82) seeds were obtained from Borovsky et al 46 .…”

“…The effect of NO on regeneration was done by placing leaf segments on Reg medium containing NO donors (5 µM Molsidomine), and NO synthesis inhibitor (5 μM DiPhenyleneiodonium) see Subban et al 30 .…”